To enhance the efficiency and accuracy of detecting systemic pesticide residues in fruits and vegetables, a microarray biochip method was utilized to screen antibody and antigen materials for six systemic pesticides: carbendazim, carbofuran, acetamiprid, procymidone, fipronil, and clothianidin. A microarray biochip was prepared, and a rapid biochip-based detection method was established for simultaneous quantitative determination of these six pesticide residues in fruit and vegetable samples. The sensitivity and accuracy of the developed method were evaluated across six types of fruits and vegetables, namely, garlic chives, leaf lettuce, tomato, sweet orange, pear, and grape. The linear correlation coefficients were >0.99 for the six pesticides within the following mass concentration ranges: carbendazim (2~32 μg•L-1), carbofuran (1~16 μg•L-1), acetamiprid (2~32 μg•L-1), procymidone (15~240 μg•L-1), fipronil (1~16 μg•L-1), and clothianidin (0.5~8 μg•L-1). The limits of detection and quantification for all pesticides in the six matrices were below half the maximum residue limits specified in national standards. Spike recovery rates ranged from 74.55% to 121.69%, with relative standard deviations (RSDs) below 14.13%. A comparison with confirmatory methods using actual positive samples showed RSDs of 5.32~15.98% between the two methods, indicating that the microarray biochip method possessed excellent sensitivity, accuracy, and consistency with confirmatory methods. The simplicity, high efficiency, and high throughput of this approach demonstrate broad application potential in agricultural production and market circulation. Overall, these findings provide a theoretical basis for developing rapid detection methods for systemic pesticide residues in fruits and vegetables.